Biopsy method

ABSTRACT

THIS PATENT DISCRIBES A NOVEL METHOD OF RAPIDLY PREPARTING TISSUE FOR BIOPIC EXAMINATIONS WHICH PROVIDES FOR THE RAPID PREPARATION OF THINNER TISSUE SECTIONS, SAID METHOD COMPRISING INFILTRATING A FIXED AND DEHYDRATED TISSUE SAMPLE WITH A LIQUID ULTRAVIOLET LIGHT-CURABLE THERMOSETTING RESIN SYSTEM HAVING A CURING THRESHOLD IN THE PRESENCE OF ULTRAVIOLET LIGHT ON THE ORDER OF ONE MINUTE OR LESS, PREFERABLY COMPRISING A POLYESTER CONTAINIG CARBON-TO-CARBON UNSATURATION, AN ETHYLENICALLY UNSATURATED MONOMER POLYMERIZABLE THEREWITH, AND AN ACTIVATOR, AND CURING SAID SAMPLE IN THE PRESENCE OF ULTRAVIOLET LIGHT FOR SEVERAL MINUTES TO FORM A RIGID BLOCK ADAPTED TO YIELD ULTRATHIN SECTIONS. THE METHOD OF THIS INVENTION IS PREFERABLY CONCERNED WITH THE USE OF A THERMOSETTING RESIN SYSTEM WHICH GIVES A CLEAR OR OPTICALLY TRANSPARENT POLYMER WITHIN THE RIGID BLOCK, CAN BE SLICED INTO SECTIONS HAVING A THICKNESS OF AS LITTLE AS 0.2 MICRON IN SMALL SECTIONS AND A CORRESPONDING IMPROVEMENT IN LARGE SECTIONS.

United States ce- 3,679,450 Patented July 25, 1972 BEST AVAILABLE COPY 3,679,450 BIOPSY METHOD Leroy Earl Beightol, Los Angeles, Calif., assignor to Roy Nutt No Drawing. Filed Mar. 16, 1970, Ser. No. 20,114 Int. Cl. A01n 1/00 US. Cl. 117-3 13 Claims ABSTRACT OF THE DISCLOSURE This patent describes a novel method of rapidly preparing tissue for biopsic examinations which provides for the rapid preparation of thinner tissue sections, said method comprising infiltrating a fixed and dehydrated tissue sample with a liquid ultraviolet light-curable thermosetting resin system having a curing threshold in the presence of ultraviolet light on the order of one minute or less, preferably comprising a polyester containing carbon-to-carbon unsaturation, an ethylenically unsaturated monomer polymerizable therewith, and an activator, and curing said sample in the presence of ultraviolet light for several minutes to form a rigid block adapted to yield ultrathin sections. The method of this invention is preferably concerned with the use of a thermosetting resin system which gives a clear or optically transparent polymer within the rigid block, can be sliced into sections having a thickness of as little as 0.2 micron in small sections and a corresponding improvement in larger sections.

BACKGROUND OF THE INVENTION The standard technique used to prepare tissue sections for biopsic examination is the paraflin block method. This method has been developed to a fine detail and involves immersing a tissue sample in 70% alcohol for 16 hours, 85% alcohol for 8 hours, 95% alcohol for 16 hours, and 100% alcohol for 2 hours to dehydrate the tissue. The dehydrated tissue is then immersed in a 100% alcohol and benzene mixture for one hour, pure benzene for one-half hour, and fresh benzene for another half-hour to prepare the tissue for penetration by the parafiin. The benzene saturated tissue is then placed in molten parafiin at about 60 C. for one-half hour, followed by three one-half hour immersions in fresh parafiin before it is placed in a block containing fresh parafiin and allowed to cool and harden preparatory to the cutting of sections. The cutting of sections is performed on a microtome. After cutting the sections, the paraifin is removed by passing the sections through several short immersions in benzene and then alcohol. The sections are then immersed in water, stained and mounted on slides for examination. The overall process described requires a total of at least 16 hours. This process is described in Lilhe, Histopathology Technique.

The parafiin block method, while familiar to those skilled in the art, and capable of practice using readily available equipment and instruments, is characterized by several major problems. One major problem is that a tissue sample prepared by the parafiin block method cannot be sliced on the microtome into sections of less than about 2 to 4 microns thickness. In general, thinner sections facilitate the biopsic examination. Because of the thickness of sections obtained by the paraflin block method, this procedure does not differentiate certain types of cells.

More recently, it has been proposed to use various plastic materials such as nitrocellulose and polymethyl methacrylate in lieu of parafiin. The procedures proposed have been very slow, requiring several hours or more to yield a rigid block in the presence of ultraviolet light. Further, these materials have proven to be unsuccessful in other respects. The best consistent results reported are tissue sections having a thickness of one micron, there also being a report of a tissue section of 0.5 micron being obtained with a methacrylate infiltrant and special care on a piece of tissue the size of a renal needle biopsy.

The present invention is believed to represent a significant advance in the art by providing for biopsic sections in a rapid manner which are thiner for any given area of section than are attainable by procedures previously developed.

SUMMARY OF THE INVENTION Briefly, the present invention comprehends a novel method of rapidly preparing tissue for biopsic examination which comprises infiltrating a tissue sample with a liquid ultraviolet light-curable thermosetting resin system having a curing threshold in the presence of ultraviolet on the order of one minute or less, and curing said sample in the presence of ultraviolet light for several minutes to form a rigid block adapted to yield ultrathin sections.

The curing threshold referred to herein and in the claims means that point in time at which the liquid resin system first begins to perceptably thicken, as determined by direct visual observation. The curing threshold of the liquid resin systems used in this invention is generally from about 10 to 60 seconds. These liquid systems usually require a total exposure to ultraviolet of from about 2 to about 5 or 10 minutes, and more typically, from 2 to 3 minutes, to become rigid.

The resin preferably is clear and optically transparent, and is based on an unsaturated polyester containing carbon-to-carbon unsaturation, an ethylenically unsaturated monomer polymerizable therewith during the curing process, and an activator. The ultrathin character and the optical clarity of the sections dispenses with the need to remove the rigidifying material after sectioning and prior to biopsic examination, thereby reducing the time required for the preparation of tissue sections.

The minimum thickness of the tissue section attainable by the practice of this invention is generally less than previously possible. The minimum thickness depends on the size or area of the section, and in the present invention sections of small size can be made having a thickness of as little as 0.2 micron. For larger sections, thicknesses of 1 or 2 microns can generally be obtained.

It is an object of this invention to provide a novel method of rapidly preparing tissue sections for biopsic examination.

More particularly, it is an object of my invention to make available thinner sections than previously attainable.

Still another object of this invention is the use of optically clear infiltrant materials which need not be removed prior to the biopsic analysis.

A specific object of the invention is the use of unsaturated polyester systems which are rapidly curable in the presence of ultraviolet light in the preparation of biopsic sections.

These and other objects and advantages of this invention will be evident from the more detailed description which follows.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Conventional fixation procedures can be used such as butfered'Zenker-formol, Bouin's solution, paraformaldehyde, percent formalin, glutaraldehyde and osmium tetroxide. The length of time of fixation is varied according to the thickness of the tissue. Most tissues are fixed in Zenker-formol from one to three hours: Tissues are normally trimmed to a few tenths of a cm. in thickness to permit better fixation and more rapid dehydration and infiltration of the plastic mixture. The fixative can be removed by washing in cold, running tap water for to 45 minutes, depending on the size or thickness of tissue.

Tissues are dehydrated through 75 percent, 95 percent, and absolute ethanol for at least to 40 minutes, depending upon the thickness of the specimens. Final dehydration is accomplished for 10 minutes in anhydrous acetone. The tissues are stored in 75 percent alcohol if immediate embedding was impractical.

The following example is presented solely to illustrate the invention and should not be regarded as limiting in any way. In the example, the parts and percentages are by weight.

Example A tissue sample of about 0.2 centimeter in thickness was fixed and dehydrated as described above. The sample was then infiltrated by placing the sample in the following solution:

Wt. percent Unsaturated polyester phthalic anhydride, maleic anhydride and propylene glycol 28 Vinyl toluene 22 Benzoin (activator) 1 Isopropyl alcohol 9 Methyl methacrylate 40 The infiltrated sample is then cured by exposure to ultraviolet light (Parr 38 mercury vapor lamp, 3650 A., 150 watt) for about 2 to 3 minutes. A rigid tissue block is obtained. The tissue block is then inserted into the microtome chuck. The block may be sectionedwith any conventional microtome while the block and the knife are moistened with alcohol. The tissue sections can be stained with the most routine histologic stains such as malloryamn, Gomoris silver impregnation, periodic acid Schiif, hematoxylin and eosin, muco-carmine, Best's carmine and azure B. The stained sections are examined under a microscope in a manner familiar to those skilled in the art.

The curable resins for use in this invention, by way of example and not of limitation, are those based on the mechanisms of photocatalysts in resinous polymerization such as are obtained by photocatalytic initiation in materials having ethylenic-type unsaturation. In these materials, the initiation is of 'first order by double bond activation, and any terminal double bonds are progressively activated. In general, the initiation is proportional to the concentration of monomer, and to the square root of light intensity.

4 5 to 50% by weight of the polyester, along with about 0.002% to 5% of the photoinitiator.

Suitable photochemical initiators, sometimes also referred to in the art as photo catalysts, for use in the ultraviolet curable resin systems of my invention include, but are not limited to, benzoin, azobisisobutyronitrile, 2,2-dihydroxy-4,4-dimethoxybenzophenone, 2,2 dihydioxybenzophenone and similar materials. For example, the addition of benzoin in the amounts as low as 0.5% to monomeric styrene will not afiect the shelf life appreciably if kept away from light. Upon exposure to moderate intensity ultraviolet light, however, therate of polymerization is increased by a factor approximately 10 over styrene containing no photoinitiator.

Any light with high enough energy per quantum or of short enough wavelength can initiate polymerization directly. For practical reasons concerned with readily available energy sources, and to provide quick polymerization, it is desirable to use a photochemical initiator which acts as an absorber, and releases free radicals from ultraviolet light in the region of 3600 angstroms. Convenient spectral sources containing bands of 3600 angstrom light energy are mercury vapor discharge lamps, sunlamps, fluorescent lamps with special phosphors, and sunlight.

Having fully described the invention, it is intended that it be limited only by the lawful scope of the appended claims.

I claim:

1. A novel method of rapidly preparing tissue for biopsic examination which comprises infiltrating a tissue sample with a liquid ultraviolet light-curable thermosetting resin system comprising an unsaturated polyester containing carbon-to-carbon unsaturation, an ethylenically unsaturated monomer polymerizable therewith, and an activator, and having a curing threshold of less than one minute in the presence of ultraviolet light, curing said sample in the presence of ultraviolet light to form a rigid block adapted to yield ultrathin sections and sectioning said cured sample to form ultrathin sections.

2. A novel method of rapidly preparing tissue for biopsic examination which comprises infiltrating a tissue sample with a liquid ultraviolet light-curable optically clear thermosetting resin system comprising an unsaturated polyester containing carbon-to-carbon unsaturation, and ethylenically unsaturated monomer polymerizable therewith, and an activator, and having a curing threshold of less than one minute in the presence of ultraviolet light, curing said sample in the presence of ultraviolet light for from 2 to about 10 minutes to form a rigid block adapted to yield ultrathin sections and sectioning said cured sample to form ultrathin sections.

3. The method of claim 1 wherein the sample is initially 5. The method of claim 1 wherein the sample is subsequently sectioned in a microtome to form ultrathin sections.

6. The method of claim 2 wherein the sample is subsequently sectioned in a microtome to form ultrathin sections.

7. The method of claim 5 wherein the sections are subsequently stained.

8. The method of claim 6 wherein the sections are subsequently stained.

9. The method of claim 1 wherein the resin system has the following composition:

Wt. percent Unsaturated polyester phthalic anhydride, maleic anhydride and propylene glycol 28 Vinyl toluene I 22 Benzoin (activator) 1 'Isopropyl alcohol I 9 Methyl methacrylate 40 10. A novel method of rapidly preparing tissue for biopsic examination which comprises infiltrating a tissue sample with a liquid ultraviolet light-curable optically clear thermosetting polyester resin system comprising an unsaturated polyester containing carbon-to-carbon unsaturation, an ethylenically unsaturated monomer polymerizable therewith, and an activator, and having a curing threshold of less than one minute in the presence of ultraviolet light, curing said sample in the presence of ultraviolet light to form a rigid block adapted to yield ultrathin sections and sectioning said cured sample to form ultrathin sections.

11. The method of claim 10 wherein the sample is 15 References Cited UNITED STATES PATENTS 2,300,495 11/ 1942 Gerhart 117--3 X 2,645,618 7/1953 Ferrari 8-94.11 X 3,527,863 9/1970 Weichselbaum 424-3 2,297,351 9/ 1942 Gerhart 204-15922 2,393,580 1/ 1946 Weiskopf 424-3 3,428,470 2/1969 Welsh et al 8-94.11 X 3,496,129 2/ 1970 Wismer et al. 11793.31 UX 3,250,642 5/1966 Parasacco et al. 11793.31 3,546,002 12/1970 Radlove et al. 11793.31

OTHER REFERENCES Brenner, Commercial Implications of Radiation Modern Plastics, April 1968, pp. 116-120, 117 and 118.

WILLIAM D. MARTIN, Primary Examiner H. J. GWINNELL, Assistant Examiner US. Cl. X.R. 

